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A Modified Co-Culture System for Understanding Granulosa-Theca Cell Interactions in the Bovine Ovary
Published on: September 19, 2025
Mancozeb impairs the ultrastructure of mouse granulosa cells in a dose-dependent manner
Maria Grazia Palmerini1, Manuel Belli1, Stefania Annarita Nottola2
1Department of Life, Health and Environmental Sciences, University of L'Aquila, 67100 L'Aquila, Italy.
Abstract:
Mancozeb, an ethylene bis-dithiocarbamate, is widely used as a fungicide and exerts reproductive toxicity in vivo and in vitro in mouse oocytes by altering spindle morphology and impairing the ability to fertilize. Mancozeb also induces a premalignant status in mouse granulosa cells (GCs) cultured in vitro, as indicated by decreased p53 expression and tenuous oxidative stress. However, the presence and extent of ultrastructural alterations induced by mancozeb on GCs in vitro have not yet been reported. Using an in vitro model of reproductive toxicity, comprising parietal GCs from mouse antral follicles cultured with increasing concentrations of mancozeb (0.001-1 µg/ml), we sought to ascertain the in vitro ultrastructural cell toxicity by means of transmission (TEM) and scanning (SEM) electron microscopy. The results showed a dose-dependent toxicity of mancozeb on mouse GCs. Ultrastructural data showed intercellular contact alterations, nuclear membrane irregularities, and chromatin marginalization at lower concentrations, and showed chromatin condensation, membrane blebbing, and cytoplasmic vacuolization at higher concentrations. Morphometric analysis evidenced a reduction of mitochondrial length in GCs exposed to mancozeb 0.01-1 µg/ml and a dose-dependent increase of vacuole dimension. In conclusion, mancozeb induced dose-dependent toxicity against GCs in vitro, including ultrastructural signs of cell degeneration compatible with apoptosis, likely due to the toxic breakdown product ethylenethiourea. These alterations may represent a major cause of reduced/delayed/missed oocyte maturation in cases of infertility associated with exposure to pesticides.
Insights
Mancozeb fungicide causes reproductive toxicity by damaging mouse granulosa cells (GCs) and oocytes. This study reveals dose-dependent ultrastructural damage in GCs, potentially explaining pesticide-induced infertility.
Area of Science:
- Reproductive Toxicology
- Cell Biology
- Environmental Health
Background:
- Mancozeb, a widely used fungicide, is known to cause reproductive toxicity in mouse oocytes.
- Previous studies indicated that mancozeb induces a premalignant status in mouse granulosa cells (GCs) with decreased p53 expression and oxidative stress.
- The ultrastructural effects of mancozeb on GCs in vitro remained largely unreported.
Purpose of the Study:
- To investigate the dose-dependent ultrastructural toxicity of mancozeb on mouse granulosa cells (GCs) in vitro.
- To characterize the specific cellular alterations induced by mancozeb exposure using advanced microscopy techniques.
Main Methods:
- Utilized an in vitro model with mouse parietal GCs from antral follicles.
- Exposed GCs to increasing concentrations of mancozeb (0.001-1 µg/ml).
- Employed transmission electron microscopy (TEM) and scanning electron microscopy (SEM) for ultrastructural analysis and morphometric measurements.
Main Results:
- Mancozeb demonstrated dose-dependent toxicity against mouse GCs in vitro.
- Observed alterations included intercellular contact changes, nuclear membrane irregularities, and chromatin marginalization at lower concentrations.
- Higher concentrations led to chromatin condensation, membrane blebbing, cytoplasmic vacuolization, reduced mitochondrial length, and increased vacuole size.
Conclusions:
- Mancozeb induces significant dose-dependent ultrastructural damage in GCs, indicative of cell degeneration and apoptosis.
- These alterations are likely mediated by its toxic breakdown product, ethylenethiourea.
- GC damage may contribute to impaired oocyte maturation and pesticide-associated infertility.
